Abstract: Lotus (Nelumbonucifera), a perennial aquatic herb has been cultivated for a long time in China for not only ornamental but also medicinal and edible purposes. One unique feature of lotus leaf is the Lotus Leaf Effect (LLE), which is characterized by its perfect water blocking function and self-cleaning function. It is known that LLE stems from the special surface structure of the lotus leaf, while wax compositions and their deposition patterns on the lotus leaf surface form the chemical basis of LLE. Thus, studies elucidating the chemical basis of the LLE has great potential for multiple practical applications. Nevertheless, previous studies revealed waxy chemical compositions on the surface of lotus leaves, but hardly mentioned the detailed detection methods, let alone the in-depth analyses of homologs and isomers. In this study, a method of gas chromatography-mass spectrometry (GC-MS) was established for the determination of wax components on lotus leaf surface, which was approved to be effective in the identification of the structure and contents of various wax molecules, homologues and isomers of lotus leaf surface. In total, 18 homologues and isomers each of long-chain straight-chain diols and long-chain secondary alcohols, 6 primary alcohols, and several fatty acids and alkanes were identified in the extract of lotus leaf surface. Among them, the homologues and isomers of C27-C35 long-chain straight-chain diols and C27-C38 long-chain straight-chain secondary alcohols accounted for 71% and 20% of the total wax contents, respectively, while C29 diols accounted for 87% of the total long-chain straight-chain diols and C29 secondary alcohols accounted for 86% of long-chain straight-chain secondary alcohols, respectively. Altogether, the results provide not only a future study direction on regulatory mechanisms of lotus leaf wax, but also a technical support for the potential application of LLE such as in hydrophobic, bionic and textile materials.